Pyrethrum plane named Arizona

ABSTRACT

A new and distinct cultivar of Chrysanthemum cinerariaefolium known by the cultivar name Arizona, and particularly characterized by the combined characteristics of high pyrethrin content, environmental stress tolerance, uniform flower stalk extension and substantially erect growth.

BACKGROUND OF THE INVENTION

The present invention comprises a new and distinct variety ofChrysanthemum cinerariaefolium, which has been designated by thecultivar name Arizona. Chrysanthemum cinerariaefolium, Vis. Arizona isthe product of selective breeding program having the objective ofcreating new Chrysanthemum cultivars whose characteristics includeenvironmental stress tolerance, high pyrethrin content, uniform flowerstalk extension and substantially erect growth. The new Chrysanthemumplant is largely sterile. Chrysanthemum cinerariaefolium Vis. Arizona isthe result of a recurrent selection breeding program started in 1979from Chrysanthemum cinerariaefolium seed of unknown origin. The breederstock is held by the University of Arizona, College of Agriculture,Department of Plant Sciences, Tuscon, Ariz.

BREEDING METHODOLOGY

Chrysanthemum cinerariaefolium seed of unknown origin was germinated andgrown in a greenhouse. In 1980, vigorous transplants were chosen fromthe greenhouse grown nursery stock and transplanted to a field at theUniversity of Arizona, Marana Agricultural Center. Over 99% of thetransplants failed to survive the either the high summer temperatures(daytime temperatures) ranges from about 95° F. to about 115° F.),exhibiting crown rots associated with high night temperatures (rangingfrom about 80° F. to about 100° F.), the sub-freezing winter nights andthe need to exhibit a measure of drought tolerance between scheduledirrigations. Some of the survivors set seed which was outcrossed asChrysanthemum cinerariaefolium was self-incompatible.

The seed from the surviving plants was harvested, germinated in a growthchamber, and transplanted to the greenhouse. The young plants weretransplanted to an adjacent field area to establish a second field testand grown in 24" row spacings on cotton beds with 40" centers. Afterover one year in the field, the best of the second generation plantswere split into several pieces. These asexual propagules were planted inrows and used to evaluate the phenotypes of the plants. Phenotypes whichexhibited a planar flowering habit by flowering at the top of the plant,lodging resistance and ease of picking were selected. All breeding lineswere evaluated for pyrethrin content and quality, determined by therelative proportion of the six distinct chemical components ofpyrethrin.

Likely, due to the extreme climatic stresses at the Marana field, someof the clones showed a biannual tendency. Because of this and otherfield conditions at that location which made continued growthundesirable, the most promising asexual propagules, their seed and someseedling plants were then transplanted over a period of time to theUniversity of Arizona Campus Agricultural Center in 1983 in threeadjacent field areas A, B and C. In 1984 and 1985 the best of thesematerials, sister lines grown from seed and asexual propagules from thegreenhouse were transplanted to a new field area D. Phenotypes andpyrethrin content and quality were evaluated annually.

To test the stress tolerance of the plants to high temperatures andsalinated soil, in 1981 seedlings from the greenhouse were transplanteda field plot was established at the University of Arizona, SaffordAgricultural Center. These clones were irrigated with salinated waterwhich resulted in soil salinity levels approaching 1/3 that of seawater. Survival rates of these transplants were extremely low and theplants were badly stunted. Pyrethrin levels were lower than desired.

All surviving clones from the Safford field were transplanted back tothe University of Arizona Campus Agricultural Center in 1985 in a newfield area E, and seed from the most promising "salt tolerant" cloneswere replicated in 30 foot half sib progeny tests.

At the same time 30 rows were planted in a new field area F at theUniversity of Arizona Campus Agricultural Center. Each of these thirtyrows were divided into seven, thirty foot sections. Seed from the mostpromising clones growing in field areas A through D were sownhead-to-row in randomized duplicate replications within field area F.Field area F was divided into three subplots with randomizations withineach sub-plot. Rows 1 through 6 were half sib progeny of clones whichdisplayed excellent lodging resistance, but on which pyrethrin contentdata were imcomplete at planting time. Rows 7 through 21 comprised halfsib progeny whose parent clones exhibited good lodging resistance,planar flowering habit and a relatively high pyrethrin content comparedto the base population. Rows 22 through 29 were half sib progeny ofother promising clones, and seed from all other promising clones as wellas four entries comprising a composite "bulk" of seed from all field Fentries. After one season, the three top pyrethrin producing plants fromfield area D were split and established in 30 foot areas of field area Fto enable evaluation of pyrethrin levels from genetically identicalclones.

The breeding program has produced three distinct germplasms, one ofwhich was asexually propagated and is referred to as Clone CA 87 F4-101. This clone was originally identified in a high pyrethrinproducing clonal progeny in the field at the University of Arizona,Marana Agricultural Center. The progenitors of Clone CA 87 F 4-101 weremaintained by recurrent selection for high pyrethrin content, planarflowering habit, lodging resistance and ease of picking in the originaltest area at the University of Arizona, Campus Agricultural Center. Thisclone is the asexual propagule of a single plant (identified as CA 85 D7-54) which was in turn selected from the half sib progeny of a plantidentified as CA 83 A 7-13. Seven daughter clones (identified as CA 87 F4-101; 102; 103; 104; 105; 106 and 107) of the single parent plant havebeen evaluated for agronomic phenotype consistency and pyrethrin contentand quality.

PYRETHRIN ANALYSIS METHODOLOGY

Flowers are harvested in April at the 1/2 to 3/4 disc floret opendevelopmental stage. Flower samples are randomly collected and harvestedonto ice and stored in darkness until transported from the field to thelaboratory. Flower samples are then counted, weighed and stored at -70°C. The flowers are freeze dried in a lyophilizer for at least 24 hours,after which dry weights are taken. Flowers are then ground for 20 to 25seconds in a grinder yielding a somewhat coarse yellow powder. Groundsamples then are stored at -70° C. until extracted.

For pyrethrin extraction, 0.2 g of the ground flowers are added to a 50ml culture tube, 10 ml of spectograde hexane is added to the groundflowers and the tubes agitated slowly on a rotator for at least tenminutes. The tube is then emptied into a miracloth square and decantedinto a second 50 ml tube. The ground flower cake is returned to theoriginal tube, another 5 ml of hexane is added and the sample is againmixed on the rotator. The contents are again decanted into a miraclothsquare and the flower cake squeezed manually to expel all solvent intothe second tube combining it with the first washing. The final volume ofhexane flower extract is adjusted to 12 ml by evaporation undernitrogen, or by adding additional hexane. 3 ml of extract is pipettedinto a serum collected vaccutainer tube and evaporated to dryness undernitrogen. The sample is stored under refrigeration until needed.

The evaporated sample residue is redissolved in 3 ml of HPLC grademethanol by vortexing for 30 seconds. The dissolved sample is filteredthrough a Gelman 0.45μ Acrodisc syringe into a 5 ml culture tube andstored in a light-proof box under refrigeration until assayed.

Quantitative analysis of the pyrethrin content of the samples arecarried out using a Varex Rosa-1 autosampler and injector interfacedwith a Beckman dual pump 421A controlled, model 165 variable wavelengthdetector high performance liquid chromatograph, linked with a Beckman427 microprocessor-controlled integrator. An Upchurch Scientificpre-column filled with Altech C-18 pre-column packing was mounted aheadof a Beckman Ultrasphere C-8 (or C-18) analytical column. The wavelengthutilized was 229 nm, which was determined to be optimum to resolve themajor pyrethrin components, based upon analysis of extinctioncoefficients of each of the six pyrethrin components across an array ofwavelengths. The range of the instrument was set at 0.2AUFS.

At the time of sample injection, the dual pump system was programmed todeliver a 50/50 HPLC grade acetonitrile/double distilled, degassed waterproportion. Two minutes after injection of the sample through a 10 μlloop, the gradient was programmed to increase at the rate of 1 and 2/3%acetonitrile per minute, for six minutes. The rate of change is thendecreased to 0.93% acetonitrile per minute for 25 minutes. All pyrethrinpeaks elute within 30 minutes. At the end of a 33 minute run, theacetonitrile is at 73.25%. A clean out step of 50/50 acetonitrile isprogrammed for 10 minutes between each sample injection. Time betweenautomatic sample injections is 46 minutes.

All six pyrethrin components, i.e. Pyrethrin I, Pyrethrin II, Cinerin I,Cinerin II, Jasmolin I and Jasmolin II, are resolved as separate peaks,electronically integrated and expressed as area units at a givenretention time (RT). Such integrations are highly repeatable over theseveral week period necessary for analysis of a years flower sampledata. One or more standard pyrethrin samples is injected every fewsamples, and the integrated area of the individual pyrethrin componentsof this industry analyzed sample (Johnson Wax, East African Kenyan Boardanalyzed standard mixture 304) are used to quantify pyrethrins inArizona grown clones.

Flowers of the daughter clones exhibit superior pyrethrin content whencompared with the majority of other clones tested. High performanceliquid chromatography analyses of hexane extracted, freeze dried flowersare presented in Table 1. The pyrethrin content of the clones, asmeasured by levels of Pyrethrin I (Chrysanthemum-monocarboxylic acidhaving the formula C₂₁ H₂₈ O₃ or the ester thereof), Pyrethrin II(Chrysanthemum-dicarboxylic acid having the formula C₂₂ H₂₈ O₅ or theester thereof), Cinerin I, Cinerin II, Jasmolin I, Jasmolin II and thePyrethrin I/Pyrethrin II ratios, which meet or exceed that of theAuthentic Kenyan pyrethrin standard. Table 2 presents agronomiccharacteristics of CA 85 D7-54 daughter clones which indicate markedlyconsistent plant height, flower diameter and flower weight between thedaughter clones.

                  TABLE 1                                                         ______________________________________                                        Peak areas at 229 nm × 10.sup.1                                                         Authentic      Clone      Clone                                               Kenyan         CA 87      CA 87                               Compared RT.sup.1                                                                             STD 304   RT   F 4-103                                                                             RT   F 4-106                             ______________________________________                                        Cinerin II                                                                             18.9   70.9      18.7 39.8  18.7 37.5                                Pyrethrin II                                                                           19.6   468.7     19.4 357.9 19.4 438.1                               Jasmolin II                                                                            22.7   39.5      22.5 21.4  22.4 28                                  Cinerin I                                                                              28.8   73.9      28.5 47.6  28.4 46                                  Pyrethrin I                                                                            29.3   693.7     29   621.4 28.9 751.2                               Jasmolin I                                                                             33     29        32.8 29    30.0 37                                  Py I/Py II Ratio.sup.2                                                                    1.48             1.74       1.71                                  ______________________________________                                         .sup.1 RT = Retention Time (Min.)                                             .sup.2 Not corrected for molar extinction coefficient differences        

                  TABLE 2                                                         ______________________________________                                                  Fresh     Dry       Flower Head                                                                            Plant                                            wt./g/100 wt./g/100 diameter height.sup.2                           Clone I.D..sup.1                                                                        flowers   flowers   mm       cm                                     ______________________________________                                        CA 88 F 4-103                                                                           96        20        14.2     81                                     CA 88 F 4-104                                                                           82        19        14.2     85                                     CA 88 F 4-106                                                                           121       26        15.7     81                                     CA 88 F 4-107                                                                           85        27        15.2     84                                     ______________________________________                                         .sup.1 Clones are daughter clones of CA 85 D754 and were harvested on         different dates accounting for some morphological variance.                   .sup.2 Clones differed in overall crown size, also accounting for some        morphological variance.                                                  

It has been found that the balance of pyrethrin isomers is under verystrict genetic control and serves as a unique molecular fingerprint ofeach individual pyrethrin clone. This characteristic is readilydiscernable is asexually propagated plant material. Clone CA 87 F 4-101was found to be sterile and did not set viable seed even whenpollinators were present. Thus, propagation is possible only throughasexual means, and the daughter clones of CA87 F 4-101 were allidentical.

The accompanying photographic drawings show typical inflorescence andfoilage characteristics of CA 87 F 4-101 with the colors being as trueas possible with such type of illustrations.

FIG. 1 shows a row of the selected clone "Arizona" (CA 87 F 4-101)marked with a red flag, alongside rows of half sibs; and illustratesrows of half sibs; and illustrates the earlier, more profuse and uniformblooming, erect flower stems and uniform height as compared to half sibclones depicted.

FIG. 2 illustrates a pressed herbarium specimen designated Chrysanthemumcinerariaefolium Vis Arizona, showing a single mature flowering stem andtypical leaves of the claimed variety sectioned for convenience inpressing.

FIG. 3 illustrates a close-up of a single normal outcrossing fertileclone, which shows a halo of open florets noticeably raised above thesurface of both the disc and ray florets such as that disclosed in U.S.Plant Pat. No. 5,848 issued Jan 6, 1987 to Bhat et al. entitled"Chrysanthemum Plant named Hypy".

FIG. 4 illustrates a typical flower of CA 87 F 4-101 at the samedevelopmental stage as that illustrated in FIG. 3, emphasizing the fullydeveloped wide-open florets characteristic of this sterile clone.

FIG. 5 illustrates a vertical section through the middle of the flowershowing the shape of the receptacle and lengths of the ovary, tubulardisk floret and pedals of the ray florets.

FIG. 6 illustrates a typical flower of Chrysanthemum cinerariaefoliumVis Arizona showing the florets and petals characteristic of this clone.

Clone CA 87 F 4-101 has very large flowers, prolific flowering andexcellent vigor which correlate with good lodging resistance. Incomparison with a normal, outcrossing fertile clone, which shows a haloof open florets noticeably raised above the surface of both the disc andray florets, a typical flower of CA 87 F 4-101 at the same developmentalstage exhibits a lack of developed open florets. (FIGS. 3 and 4). Thisclone blooms synchronously, and averaged 300-400 flowers per clone.However, mature clones of fertile, sister lines have averagedapproximately 800 flowers per plant. Pyrethrin analysis of this clone, asterile sister clone (designated F 4-117) and Kenyan Standard 304, showpyrethrin content of about 2% or greater as set forth in Table 3.

                  TABLE 3                                                         ______________________________________                                        Year Genotype     Py I/Py II Ratio                                                                           Percent Pyrethrins                             ______________________________________                                        1986 Kenyan Std. 304                                                                            1.42         2.00                                                D 7-54       1.18         2.09                                                D 7-19       1.33         1.83                                           1987 Kenyan Std. 304                                                                            1.42         2.00                                                F 4-101      1.90         2.03                                                F 4-117.sup.1                                                                              n/a          n/a                                            1988 Kenyan Std. 304                                                                            1.36         2.00                                                F 4-101      1.56         1.95                                                F 4-117      1.61         2.25                                           ______________________________________                                         .sup.1 Flowers from Clone F117 were not picked in 1987 due to small crown     size.                                                                    

Typical flowers of the clone contain considerably less pollen than theusual flower. Anthers contain what appear to be grayish, incompletelymatured pollen grains. Some pollen looks fully developed and cansometimes be observed in a few anthers, depending upon the environment.Florets of the clone open more rapidly towards the center than the usualflowers. FIGS. 3 and 4 show flowers at the same relative stage ofdevelopment; the flower of the clone has completely opened florets,whereas the flower of the other lines have only partially opened floretsat the same stage of development. The clone is characterized by widelyopen florets, which are typical of sterile or partly sterile flowersfrom many species. The ray florets of the clone are believed to becompletely sterile and the florets of the clone rapidly open fromoutside toward center. Two separate field plantings of seed from theclone have failed to germinate, whereas adjacent plantings of seed fromother plants succeeded.

Flowers of the clone have a strong scent which has been described byobservers as an intense musty, aromatic chemical smell. Splitting theflower in half greatly intensifies the scent, so it does not appear tobe a nectar volatile aroma. Flowers typically display from 22 to 27pedals, compared to 19 to 22 of the typical pyrethrum flower. The clonehas thicker stems which provide visually greater lodging resistance thanother plants of similar height. Flowering stems of every other plantwhich are 90% as tall or taller than the clone fall over under fieldconditions. Peak bloom dates were Apr. 25, 1989, Apr. 29, 1988 and Apr.28, 1987. Plants heights averaged over 90 cm; flowers per plant averagedover 400 (with a maximum of 500+); typical plants ranged from 50 to 80cm with an average of about 65 cm; flowers per plant ranged from 4 toapproximately 450. A typical plant flower head diameter ranged from 9 mmto 14.3 mm with a mean of about 12.8 mm. Flowers per stem of the cloneaveraged 5 (range of 1-10 with considerable variability); while theconventional plant averages 3 to 4 flowers on virtually every stem. Theclone splits readily, and the splits exhibit high field survival rates(over 90%) and a medium sized plant will usually yield 15 to 45 small tomedium size splits, each with a sturdy, untwisted root system. Allclones derived from the original plant bloomed in close synchrony,within 2 to 3 days, and reached peak flower opening rapidly, retainedpyrethrum content for at least two weeks and have agronomicallyfavorable attributes of flowers born at the same height, enabling theflowers to be mechanically harvested "once over".

The physical size of the clone relative to the great majority of otherplants with similar genetic background, including sterility, plantheight, flower diameter, pedal member, stem diameter and number offlowers per stem indicates this plant may be a triploid.

It has been found that the clone exhibits optimum growth patterns in awide range of elevations and stressful growing environments. Table 4summarizes environmental responses of Chrysanthemum cinerariaefoliumVis. Arizona across a range of Arizona environments:

                  TABLE 4                                                         ______________________________________                                        Location                                                                             Elevation (m)                                                                            Climatology and Agronomics                                  ______________________________________                                        Lakeside                                                                             2137       Snow cover protects crowns, cold/                                             dry winter kill plants.                                     Elfrida                                                                              1213       Excellent survival; plants die back to                                        crown in winter.                                            Safford                                                                              900        Plants under salt stress, perennial                                           habit, small stature, lower pyrethrin                                         levels.                                                     Tucson 714        Near optimum location, high pyre-                                             thrin levels, good survival at                                                temperatures in range of about 17° F.                                  to about 111° F.                                     Tucson 699        Good environment, high soil nitrates                                          burnt many transplanted splits.                             Marana 598        More extreme temperatures in the                                              range of about 15° F. to about 115° F.                          and high summer nighttime                                                     temperatures over 80° F. elicit crown                                  rots.                                                       Yuma    50        High summer temperatures and                                                  humidity cause fungus crown diseases                                          and kill plants.                                            ______________________________________                                    

In describing the colors, reference has been made to the book R.H.S.Colour Chart, published by The Royal Horticultural Society, London,England in association with the Flower Counsil of Holland.

INFLORESCENCE

A. Capitulum: Flat, daisy, diameter across face approximately 40-80 mm.

B. Corolla of ray florets: White, bright tonality.

C. Corolla of disk florets: Approximately orange-yellow 17A; (freshcolors); approximately yellow 7A to 13A (dried colors).

D. Reproductive organs: Male flowers reduced in number and greatlyreduced in function. Female flowers, present in disk florets, uncertainpresence in ray florets.

PLANT

A. Foliage:

Upper leaves.--Approximately green 137C to 137D.

Midplant leaves.--Green 137B to 137C.

Lower leaves.--Green 137A, 137B to 137C.

Underside of leaves.--Approximately green 147B to 147C.

It can be appreciated by those skilled in the art that a new asexuallyreproduced pyretrum plant, designated Chrysanthemum cinerariaefoliumVis. Arizona has been developed which exhibits the followingcharacteristics:

1. Stress tolerance to high heat and mild freezes.

2. Adaptation to Arizona latitude and elevation.

3. Pyrethrin content of 2% or more.

4. Balance of Pyrethrin content I to Pyrethrin II (PyI/PyII ratio) closeto that of preferred East African pyrethrins.

5. Sterility.

6. Readily asexually propagated from splits.

7. Excellent plant vigor and spring regrowth.

8. Synchronous flowering. All flowers mature at nearly the same time,all daughter clones bloom together.

9. Vigorous and profuse flowering.

10. Planar flowering habit.

11. Large flowers exhibiting goof flower form.

12. Plant color of medium green with a slight grey undertone.

13. Medium to large cut leaves.

14. Tall and erect phenotype.

15. 100 flower dry weight is about 23.0 grams.

16. Flowers are easily broken off stems, flowers normally retain no stemwhen picked.

17. Perennial habit.

18. Good lodging resistance, due to large, stiff stems.

Thus, a new and distinct cultivar of Chrysanthemum cinerariaefoliumdesignated Arizona has been described which exhibits both high pyrethrincontent and environmental stress tolerance. The cultivar Arizona may beasexually reproduced to yield propagules of like phenotypes, the uniformphenotype having the same genetic fingerprint of the six pyrethrinisomers as that of the parent clone, thereby facilitating identificationof the plant lineage.

I claim:
 1. A new and distinct cultiar of Chrysanthemum cinerariaefoliumplant known by the cultivar name Arizona, and particularly characterizedas to uniqueness as described and illustrated herein by the combinedcharacteristics of high pyrethrin content, environmental stresstolerance, uniform flower stalk extension and substantially erectgrowth.